1. Field of the Invention
This invention relates generally to chemical methods for diagnosing Cushing's syndrome in domestic animals, particularly dogs and horses. More specifically, this invention relates to methods for detecting Cushing's syndrome based upon chemical analysis of hepatic functions. Central to any such chemical analysis is an understanding of the terms Cushing's syndrome, glycogen, glycogenesis, glycogenolysis and glucocorticoids.
Cushing's syndrome. Cushing's syndrome is a disease condition caused by the excessive production of corticosteroids by the adrenal cortex. The condition is often due to tumor or hyperplasia of either the pituitary gland or the adrenal cortex itself.
Glycogen. Glycogen is the chief storage form of carbohydrate in animals and is analogous to starch in plants. The principle organ in which glycogen is stored in the body is the liver. The process of glycogen synthesis (glycogenesis), and that of its breakdown (glycogenolysis) is known to proceed by two separate pathways.
Glycogenesis. The initial reaction required for the entrance of glucose into the series of metabolic reactions which culminate in the synthesis of glycogen is phosphorylation of glucose at the C-6 position. Glucose is phosphorylated by adenosine triphosphate (ATP) in the liver by an irreversible enzymatic reaction which is catalyzed by a specific glucokinase. This undirectional phosphorylation permits the accumulation of glucose in the liver cell since the phosphorylated sugars do not pass freely in and out of the cell in contrast to the readily diffusable free sugars. The trapped glucose-6-phosphatase is converted to glucose-1-phosphatase, a reaction catalyzed by phosphoglucomutase. Glycogen is synthesized from the glucose-1-phosphate through reactions involving the formation of uridine derivatives. In the presence of polysaccharide primers and the enzyme glycogen synthetase, the glucose moiety of the urine derivatives is linked to the polysaccharide. Through repeated transfers of glucose, the polysaccharide chain is eventually lengthened until a glycogen molecule is formed.
Glycogenolysis. The breakdown of liver glycogen to glucose takes place by a second pathway. In the presence of inorganic phosphate, the glucose linkage of glycogen is successfully broken by active phosphorylases. Epinephrine and glucagon influence the phosphorolytic breakdown of glycogen to glucose. The phosphorolytic enzyme exists in the liver in two forms: an active form designated liver phosphorylase (LP) which contains phosphate and an inactive form designated dephosphorylase (dephospho-P), in which phosphate has been removed. The transformation between the active and the inactive forms are catalyzed by specific kinase enzymes. Normally the level of LP is low and the epinephrine and glucagon shifts the equilibrium toward a higher level of LP. The net result is an increased phosphorolytic breakdown of glycogen to glucose. A hyperglycemia is observed clinically following the injection of either of these two hormones.
Glucocorticoids. Glucocorticoids promote liver glycogen storage. This increase in liver glycogen storage has been attributed to glucocorticoid enhancement of gluconeogenesis, hyperglycemia, decreased glycogenolysis and decreased glucose oxidation.
Glucagon. Glucagon has been used in certain diagnostic procedures as well as in various pharmaceutical treatments. It is a polypeptide secreted by the alpha cells on the pancreas. The primary structure of porcine, bovine and human glucagon are identical. Glucagon is produced as a by-product of insulin production from pork and beef pancreases. Injections of glucagon are known to elevate blood glucose levels by causing hepatic glycogenolysis. Furthermore, it is known that under standardized conditions, glucagon induces reproducable hyperglycemia in test animals.
However, despite the knowledge of glycogenesis and glycogenolysis, it has not been heretofore fully appreciated that the intravenous administration of glucagon (glucagon tolerance test) may be used to detect the excessive storage of liver glycogen which can be diagnostic of Cushing's syndrome.
2. Prior Art
Cushing's syndrome in the dog is frequently a diagnostic challenge to the practicing veterinarian. Cushing's must be considered in any patient presented with histories of increased water consumption, urination, elevation in serum alkaline phosphatase levels or symmetrical alopecia. However, none of these signs are diagnostic for Cushing's, and in fact often are associated with many other more common clinical entities. Because the treatment for Cushing's is often associated with significant cost and occasionally high morbidity, a definitive diagnosis of Cushing's is necessary before treatment.
The clinical signs associated with Cushing's are caused by increased levels of endogenous cortisol. The current method of diagnosing Cushing's includes the ACTH stimulation test and/or the dexamethasone suppression test. The ACTH stimulation test is performed by taking blood for a baseline cortisol assay followed by the administration of ACTH and again collecting blood for cortisol assays 1-3 hours after the administration of the stimulating hormone. The Cushing's animal should have an exaggerated cortisol level following ACTH administration. The second most commonly employed test for the diagnosis of Cushing's is the dexamethasone suppression test. Again, blood samples are collected for baseline cortisol values and dexamethasone is administered. Blood is collected twice for cortisol assays, once at 3 hours and again at 8 hours. Normal animal cortisols become significantly depressed following dexamethasone therapy; Cushing's patients do not.
Both the ACTH stimulation test and dexamethasone suppression test require assays for blood cortisol. These are generally radioimmune assays and are not run in veterinarian's offices and must be packaged and mailed to specialized laboratories for analysis. These assays also are relatively expensive. In addition, these individual tests are associated with approximately 20% false negative results and therefore they are frequently combined in confirming the diagnosis of Cushing's and thereby further raising the outside lab charges. Hence, the principle disadvantages of these tests are their high cost, slow turnaround, cumbersome sample preparation procedures and false negatives. Consequently, any new test for Cushing's which is sensitive, reliable, economic and capable of in-house utilization in short time periods would be of great practical value to veterinary medicine. In order to obviate these problems, applicant has developed a safe, reliable test for diagnosis of Cushing's syndrome. Applicant's test is a heretofore unappreciated application of the glucagon tolerance test. Those skilled in the art will, of course, appreciate that variations of this test have been used to diagnose other pathologies. For example, glucagon tolerance test has been used in the dog to differentiate pancreatic tumorbearing dogs in which insulin release is stimulated by the transient hyperglycemia product following glucagon administration; blood insulin levels are then measured. See Johnson RK: Insulinoma in the dog. Vet Clin North Am 7(3): 629-635, (1977). The cat has also been used as an in vivo means of assaying small quantities of glucagon. See Behrens OK, Broner W: Glucagon, Vitamin, and Hormone, vol XVI-16: 263-301, Academic Press, New York (1958). See also, Roberts, Steven M., et al., Effect of Ophthalmic Prednisolone Acetate on the Canine Adrenal Gland and Hepatic Function, AM. J. Vet. Res., Vol. 45, No. 9 (September 1984).